Long non-coding RNA MEG3 knockdown represses airway smooth muscle cells proliferation and migration via sponging miR-143-3p/FGF9 in asthma

Background Asthma is a respiratory disease characterized by airway remodeling. We aimed to find out the role and mechanism of lncRNA MEG3 in asthma. Methods We established a cellular model of asthma by inducing human airway smooth muscle cells (HASMCs) with PDGF-BB, and detected levels of lncRNA MEG3, miR-143-3p and FGF9 in HASMCs through qRT-PCR. The functions of lncRNA MEG3 or miR-143-3p on HASMCs were explored by cell transfection. The binding sites of miR-143-3p and FGF9 were subsequently analyzed with bioinformatics software, and validated with dual-luciferase reporter assay. MTT, 5-Ethynyl-2’-deoxyuridine (EdU) assay, and Transwell were used to detect the effects of lncRNA MEG3 or miR-143-3p on proliferation and migration of HASMCs. QRT-PCR and western blot assay were used to evaluate the level of proliferation-related marker PCNA in HASMCs. Results The study found that lncRNA MEG3 negatively correlated with miR-143-3p, and miR-143-3p could directly target with FGF9. Silence of lncRNA MEG3 can suppress migration and proliferation of PDGF-BB-induced HASMCs via increasing miR-143-3p. Further mechanistic studies revealed that miR-143-3p negatively regulated FGF9 expression in HASMCs. MiR-143-3p could inhibit PDGF-BB-induced HASMCs migration and proliferation through downregulating FGF9. Conclusion LncRNA MEG3 silencing could inhibit the migration and proliferation of HASMCs through regulating miR-143-3p/FGF9 signaling axis. These results imply that lncRNA MEG3 plays a protective role against asthma.


Introduction
Asthma is the chronic respiratory disease affecting children and adults, and is characterized by inflammation of the bronchi, increased viscous secretions within the tubes, and airway remodeling [1][2][3].Clinically, asthma is manifested as cough, wheezing, chest tightness, etc., which can cause lung infection and sudden death in severe cases [3].There is currently no cure for asthma, and treatment just could control the symptoms.For example, studies have found that anti-inflammatory drugs and bronchodilators can be effective in controlling asthma [4][5][6].However, traditional medications are associated with a variety of side effects and exploring new treatments is expected to be the key to treating asthma.
Accumulating evidences suggested abnormal migration and proliferation of HASMCs were responsible for changes in ASM thickness and promoting airway remodeling [7][8][9].In addition, the mechanism of airway remodeling is related to the inflammatory-promoting factors and growth factors released by inflammatory cells in the airway [10,11].In the presence of a variety of inflammatory factors, the airway epithelium in asthmatics undergoes pathological changes, such as basement membrane thickening, glandular hypertrophy and hypertrophy of smooth muscle [12].The study found that the expression of platelet-derived growth factor BB (PDGF-BB) was obviously increased on asthmatic tissues [13].Moreover, PDGF-BB can induce proliferation and migration of HASMCs, causing airway remodeling [14,15].Therefore, inhibiting proliferation and migration of PDGF-BB-stimulated HASMCs can effectively prevent occurrence of airway remodeling, which may become an effective treatment for asthma.
Long non-coding RNAs (lncRNAs) are a type of RNAs do not encode proteins and have transcripts longer than 200 nt [16,17].LncRNAs have multiple functions, for instance acting as molecular scaffolds in the nucleus, regulating chromosome structure, or as competing endogenous RNAs (ceRNAs) in cytoplasm to promote or inhibit mRNA degradation and adsorb microRNAs (miRNAs) [18,19].In addition, lncRNAs participated in regulation of various biophysiological processes, including embryonic development, cardiac development, the immune system, and the endocrine system [20][21][22].Aberrant level of lncRNAs has been found in various of diseases, such as cardiovascular diseases, tumours and neurodegenerative diseases [23][24][25][26].Thus, measuring the expression of lncRNAs in different cells or diseases could help to understand their function, or to identify valid molecular markers.Recent studies have shown thatthe expression of lncRNA Maternally-Expressed Gene 3 (MEG3) was significantly reduced in the peripheral blood of asthma patients [27], and lncRNA MEG3 played important roles in asthma by regulating Treg/Th17 balance [28].The results indicate that lncRNA MEG3 is involved in the mechanism of asthma, while the specific roles and regulatory mechanisms remain to be further investigated.
Increasing evidences demonstrate lncRNAs could act as ceRNAs to regulate the role of miRNAs in disease [18,29].MiRNA is a class of non-coding RNAs with a length of 21-23 nucleotides, which regulating gene expression at the translational level [30,31].Research have shown miR-143-3p is the direct target of lncRNA MEG3, which could be involved in periodontitis by sponging miR-143-3p to inhibit AKT/IKK signaling pathway [32].MiR-143-3p is a tumor suppressor that affects invasion, migration and proliferation [33][34][35].MiR-143-3p was found to be obviously decreased in ASMCs of asthmatic patients and may exert a protective effect by inhibiting asthmatic airway remodeling [36].However, it is unclear whether lncRNA MEG3 plays an important role in asthma through regulating miR-143-3p.
This study aimed to explore the roles and underlying mechanism of lncRNA MEG3 in proliferation and migration of ASMCs.
Previous studies have found PDGF-BB could induce airway remodeling in asthma [15].A cellular model of asthma was set up through stimulating HASMCs with PDGF-BB (25 ng/ml; Sigma, USA) for 24 h in this study.

MTT assay for cell viability
Proliferation was measured with MTT assay as previously described [37].Briefly, seeding 1 × 10 4 cells into a 96-well plate and treated for 24 h before MTT assay.Adding 20 µl MTT solution (C0009S, Beyotime, Shanghai, China) to each well and cells were cultured for 4 h at 37 °C.Absorbance levels were measured at 570 nm with a plate reading spectrophotometer.

Transwell assay for migration
Migration was assessed with Transwell assay as previously described [38].Briefly, serum-free and 10% serumcontaining medium were added to the upper and lower layers in a 24-well Transwell chamber with a pore size of 8 μm.Cells were cultured in the upper layer for 24 h.Cells penetrating to the lower layer were subsequently fixed with 4% methanol and stained with crystal violet.Cells were counted with an inverted microscope (LEICA).

Statistical analysis
Data were statistically analyzed with GraphPad Prism 9 and were expressed as mean ± standard deviation (SD).Unpaired T-test or one-way ANOVA was used for analysis, and p < 0.05 indicated statistical significance.

LncRNA MEG3 was upregulated, whereas mir-143-3p was downregulated in PDGF-BB-induced HASMCs
To explore roles of lncRNA MEG3 and miR-143-3p in asthma, this study constructed an asthma model by treating HASMCs with PDGF-BB, and detected the levels of lncRNA MEG3 and miR-143-3p with qRT-PCR.Compared with control group, lncRNA MEG3 was remarkably upregulated in PDGF-BB-induced HASMCs, while miR-143-3p was significantly reduced in PDGF-BB-induced HASMCs (Fig. 1A and B).These results suggest lncRNA MEG3 and miR-143-3p may be involved in development of asthma.
Subsequently, cell proliferation was measured with MTT and EdU assay, cell migration was detected with Transwell, and expression of cell proliferation related gene PCNA was detected by western blot and qRT-PCR.The results revealed cell viability of HASMCs induced by PDGF-BB was significantly increased (Fig. 3C), the protein and mRNA levels of PCNA was significantly increased (Fig. 3D-E), the prolifearion of HASMCs was signifixantly enhanced (Fig. 3F anf G) and the cell migration ability was significantly improved (Fig. 3H and I).In addition, in PDGF-BB-induced HASMCs, MEG3 downregulation significantly reduced cell proliferation and cell migration, and PCNA was reduced (Fig. 3C-I).However, these changes were significantly reversed via cotransfection with miR-143-3p inhibitor.These results suggest downregulation of lncRNA MEG3 can inhibit proliferation and migration of PDGF-BB-induced HASMCs by upregulating miR-143-3p.

Expression of FGF9 in PDGF-BB-induced HASMCs
Next, we examined the levels of FGF9 in PDGF-BB-stimulated HASMCs with western blot assay and qRT-PCR.The results demonstrated that the protein and mRNA levels of FGF9 were obviously increased in PDGF-BBinduced HASMCs compared to control group (Fig. 5A  and B).These results demonstrate that the expression of FGF9 is elevated in PDGF-BB-stimulated HASMCs.
MTT results showed PDGF-BB stimulation of HASMCs significantly increased cell viability, while upregulation of miR-143-3p decreased cell viability (Fig. 7D).Western blot and qRT-PCR results indicated the expression of PCNA in PDGF-BB group was significantly increased, while the upregulation of miR-143-3p could reduce the level of PCNA in PDGF-BB-induced HASMCs (Fig. 7E-F).The results of EdU assay indicated that PDGF-BB stimulation of HASMCs significantly enhanced cell proliferation, while upregulation of miR-143-3p decreased cell proliferation (Fig. 7G and  H).Additionally, as shown in Fig. 7I and J, miR-143-3p mimic could significantly reduce cell migration ability of PDGF-BB-induced HASMCs.These effects were significantly reversed after co-transfection with FGF9-plasmid (Fig. 7D-J).These data suggest upregulation of miR-143-3p can significantly inhibit proliferation and migration by reducing FGF9.

Discussion
Asthma is a common inflammatory disease of airways that usually results in airway remodeling [1,9,40].Airway remodeling manifested by bronchial fibrosis, basement membrane thickening and increased airway smooth muscle mass [41].The study found that abnormal proliferation and migration of ASMCs were involved in development of airway remodeling [7].Therefore, modulating proliferation and migration of ASMCs may be an effective way to explore the therapy of asthma.
Evidences indicated lncRNA was the molecular marker for diagnosis of asthma and can be involved in regulating proliferation and migration of ASMCs [8,[42][43][44].For example, downregulation of lncRNA MALAT1 inhibited proliferation of ASMCs through sponging microRNA-216a [45].Recent years, lncRNA MEG3 was discovered to be a tumor suppressor gene, which can be involved in angiogenesis and tumorigenesis [46,47].LncRNA MEG3 was reported to affect proliferation and migration of prostate cancer cells through regulating miR-9-5p/QKI-5 axis [48].Furthermore, lncRNA MEG3 could participate in neuronal apoptosis in stroke via sponging miR-424-5p [49].Recent research have found lncRNA MEG3 was significantly increased in asthmatic patients [27], suggesting lncRNA MEG3 may be involved in the occurrence of asthma.The lncRNA MEG3 has been reported to regulate Treg/Th17 homeostasis in asthma patients through targeting microRNA-17 [28].However, specific roles and regulatory mechanisms of lncRNA MEG3 in asthma is still unclear.
Recent studies have identified binding sites between lncRNA MEG3 and miR-143-3p, which was involved in regulating periodontal ligament cell damage [32].However, the relationship between lncRNA MEG3 and miR-143-3p in asthma has not been reported.Our study resolved roles of lncRNA MEG3 and miR-143-3p in PDGF-BB-induced HASMCs.We proved lncRNA MEG3 could negatively regulate miR-143-3p, and silence of lncRNA MEG3 could suppress proliferation and migration of HASMCs via upregulating miR-143-3p.In addition, bioinformatics database indicated FGF9 was a direct target of miR-143-3p, and miR-143-3p was negatively correlated with FGF9.FGF9 is the member of the fibroblast growth factor family, which is involved in various pathological processes such as angiogenesis, apoptosis, and tumor growth [50].FGF9 has been found to be essential for lung development and recovery from lung tissue injury [51].Furthermore, FGF9 signaling inhibited airway smooth muscle differentiation in mouse lungs [52].These studies imply that FGF9 may play important roles in smooth muscle cells.Our research showed miR-143-3p suppressed proliferation and migration of HASMCs through reducing the expression of FGF9, indicating the key role of FGF9 in the proliferation and migration of HASMCs in asthma.FGF9 may play an important role in asthma through regulating the proliferation and migration of HASMCs.
Our research is the first to elucidate the effects and mechanisms of lncRNA MEG3 and miR-143-3p on the proliferation and migration of HASMCs in airway remodeling in asthma.However, there were also some limitations of this study.Firstly, this study was mainly based on in vitro experiments to explore the role of lncRNA MEG3 in asthma, while in vivo studies need to be further validated by animal models.Besides, the expression of lncRNA MEG3/miR-143-3p/FGF9 in asthma patients and its correlation with clinical pathological parameters also need further clarification.This study also did not further explore the effects of FGF9 alone on HASMCs and on the protective effect of lncRNA MEG3 on asthma.In addition, lncRNA MEG3, as a ceRNA, can sponge a variety of miRNAs [53], and the involvement of lncRNA MEG3 in HASMCs function through regulation of miR-143-3p/FGF9 axis is one of the mechanisms identified in this study, while other mechanisms need to be explored through further studies.In future studies, we will further investigate these issues.

Conclusions
In conclusion, our study revealed the roles of lncRNA MEG3 in PDGF-BB-induced HASMCs.Downregulation of lncRNA MEG3 could inhibit the proliferation and migration of HASMCs by regulating miR-143-3p/FGF9 signaling axis.These results suggest lncRNA MEG3 plays a protective role in asthma and may be a novel biomarker for therapy of asthma.